Up to half of all transcription termination events in bacteria rely on the RNA-dependent helicase Rho. However, the nucleic acid sequences that promote Rho-dependent termination remain poorly characterized. Defining the molecular determinants that confer Rho-dependent termination is especially important for understanding how such terminators can be regulated in response to specific signals. Here, we identify an extraordinarily long-lived pause at the site where Rho terminates transcription in the 5′-leader region of the Mg 2+ transporter gene mgtA in Salmonella enterica. We dissect the sequence elements required for prolonged pausing in the mgtA leader and establish that the remarkable longevity of this pause is required for a riboswitch to stimulate Rho-dependent termination in the mgtA leader region in response to Mg 2+ availability. Unlike Rho-dependent terminators described previously, where termination occurs at multiple pause sites, there is a single site of transcription termination directed by Rho in the mgtA leader. Our data suggest that Rho-dependent termination events that are subject to regulation may require elements distinct from those operating at constitutive Rhodependent terminators.magnesium | RNA polymerase T ranscription termination is critical for accurate expression and regulation of genes. Bacteria use two types of transcription terminators: intrinsic terminators, which dissociate transcription complexes in the absence of auxiliary proteins, and factor-dependent terminators, which require the action of the RNA-dependent helicase Rho (1). Rho is responsible for 20-50% of termination events in Escherichia coli (2, 3) and performs an array of important functions in bacteria. In addition to terminating transcription at the ends of operons (4), Rho directs transcription termination within coding regions when translation slows or stalls thereby establishing transcriptional polarity (5), silences horizontally acquired DNA (3, 6), protects the chromosome from R-loops and double-strand breaks (7-9), and suppresses pervasive antisense transcription (10).Aside from these genome-wide "housekeeping" functions, Rho has also been recruited to regulate the expression of specific genes in response to signals (11). Long-established examples include a Rho-dependent terminator that controls expression of the E. coli tnaA gene in response to tryptophan availability and terminators that regulate rho mRNA levels in response to the concentration of Rho protein (12-15). The recent identification of two riboswitches and a small regulatory RNA (sRNA) that control gene expression by modulating Rhodependent termination (11,16,17) has now refocused attention on Rho as a regulatory factor. However, it is unclear whether regulated Rho-dependent terminators differ from those performing housekeeping functions.Rho-dependent terminators consist of two elements: a Rho utilization (rut) site in the nascent RNA and a downstream sequence that induces pausing of RNA polymerase (RNAP). The ATPase activity of Rho is stimulated upon...
Synthesis of long RNA chains by multisubunit RNA polymerases (RNAPs) requires accessory proteins that help RNAP bypass numerous roadblocks it encounters along the way. These “antitermination” proteins switch the elongating RNAP into a processive state, but the molecular mechanism of this phenomena remains unknown. Bacterial antiterminator RfaH is an excellent model to address this key mechanistic question because its structure, the mechanism of recruitment and its binding site on RNAP have been determined. RfaH increases expression of the distal genes in operons that have ops DNA elements. We found that RfaH binds simultaneously to the regions of β and β′ subunits that constitutes “jaws” of RNAP, locking it in a closed conformation. Deletion of a conserved β gate loop (GL) has little effect on intrinsic properties of RNAP but completely abolishes its response to RfaH and NusG, a general paralog of RfaH. qRT‐PCR analysis of ops‐containing operon expression driven by wild‐type or ΔGL RNAP in the presence or absence of RfaH confirmed in vitro observations. Finally, we showed also that GL deletion does not compromise stimulation of Rho‐dependent termination by NusG, function that is thought to be independent from AP effect of NusG and based on physical tethering of Rho to the elongation complex. We propose that NusG‐like proteins restrict conformational mobility of RNAP, thereby locking it in a processive state.
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